During protein biosynthesis, the vitamin K-dependent blood coagulation proteins undergo post-translational processing that includes gamma-carboxylation. The mechanism by which these proteins are identified for gamma-carboxylation and specific glutamic acid residues are gamma-carboxylated is not known. These proteins are synthesized in a precursor form that includes a signal peptide and propeptide extension on the amino terminus of the mature protein. Based upon the marked sequence homology of the propeptide of the vitamin K-dependent proteins and our observation that a point mutation at residue -1 in Factor IX impairs gamma-carboxylation, we believe that the propeptide is the recognition site for the proteins associated with gamma- carboxylation. To test this hypothesis, we are developing an efficient expression system for recombinant prothrombin and Factor IX. The prothrombin and Factor IX cDNA have been introduced into mammalian host cells, including Chinese hamster ovary and HepG2 cells. The recombinant prothrombin and Factor IX are gamma-carboxylated and can be isolated by immunoaffinity chromatography. This expression system will be optimized by testing various host cells, vitamin K delivery systems, and methods of introducing the DNA coding sequences into the cells. The purified recombinant prothrombin and Factor IX will be structurally and functionally characterized. The specific role of the propeptide will be evaluated by altering specific amino acids in the propeptide by site-specific mutagenesis and by deletion of the propeptide to determine the effect of these structural changes on the gamma-carboxylation of these proteins. The prothrombin signal peptide/propeptide leader will be spliced to non-carboxylated proteins by linking the prothrombin cDNA coding for the signal peptide/propeptide with the cDNA coding for the mature non-carboxylated protein of interest, tissue plasminogen activator and antithrombin III. Carboxylation of glutamic acids in these proteins will prove that the propeptide directs gamma-carboxylation. The protein in the liver responsible for recognizing the propeptide, the K-propeptide recognition protein, will be detected using a binding assay based on a synthetic peptide analog of the propeptide, -18 to -1. This protein will be purified from liver microsomes and characterized. The relationship of this recognition protein and the gamma- carboxylase will be determined. These studies will further our basic knowledge of vitamin K-dependent gamma-carboxylation during protein synthesis and may facilitate the development of recombinant gamma-carboxylated proteins for therapeutic use.